Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein encoded by the folate hydrolase 1 (FOLH1) gene. Compared with other non-specific PET tracers, PET/CT imaging targeting PSMA has important clinical value in the diagnosis and staging of PCa. PSMA is highly expressed on the surface of PCa cells and is closely correlated with tumor grade, PSA value, and prognosis. So far, two PSMA agents (⁶⁸Ga-PSMA11 and ¹⁸F DCFPyL) have been approved by the FDA for clinical application (5). Other PSMA tracers are also commonly used in preclinical studies and clinical trials, such as ⁶⁸Ga-PSMA617 and ¹⁸F-PSMA-1007. A meta-analysis based on 37 studies with 4,790 patients was conducted by Perera et al. and showed that the overall sensitivity and specificity of ⁶⁸Ga-PSMA PET/CT for initial staging of advanced PCa were 77% and 97%, respectively (6). According to a meta-analysis by Huang, an overall pooled detection rate of 94% for ¹⁸F-PSMA-1007 was demonstrated in PCa patients (7). With a combined median maximum standard uptake value (SUVmax) of 16 (3.7-77.7) for primary prostate lesions, ¹⁸F-PSMA-1007 had positive predictive values of 0.90, 0.94, and 0.84 with the identification of lesions, regional lymph node metastases, and localized prostate tumors, respectively. With the comparison of regular CT imaging and bone scanning, the accuracy of PET imaging with PSMA as the target was 27% higher (92% vs 65%), as were sensitivity and specificity (85% vs 38%, 98% vs 91%) (8). Zhou et al. made a critical comparison of ¹⁸F-PSMA-1007 PET/CT and ¹⁸F-FDG PET/CT, which were both performed on 21 PCa patients (9). The SUVmax, mean standard uptake value (SUVmean), and tumor-to-background ratio (TBR) of ¹⁸F-PSMA-1007 PET/CT were higher than those of ¹⁸F-FDG PET/CT in the primary lesions and metastases, leading to a superior detection rate in the primary PCa lesions and more significant differentiations between benign lesions and metastases. In addition, the multifocality of primary PCa lesions was presented under the ¹⁸F-PSMA-1007 PET/CT rather ¹⁸F-FDG PET/CT, suggesting the excellent PCa lession localization the PSMA tracer provides ( Figure 1 ).

PSMA PET/CT imaging makes a contribution to the diagnosis of PCa metastases. A prospective single-center study demonstrated that PSMA PET/CT imaging has modest sensitivity (71.4%) and exceptional specificity (88.9%) in detecting pelvic lymph node involvement (10). In addition, PSMA PET/CT imaging combined with sentinel lymph node biopsy in primary-identified medium to high-risk PCa resulted in 94% accuracy in original lymph node staging of PCa. This cross-validation could increase the overall sensitivity of lymph node metastasis to 100% (11). To assess the diagnostic efficacy of PSMA imaging for PCa bone metastases, a network meta-analysis that involved 45 studies with a total of 2,843 patients and 4,263 lesions was completed by Liu et al. (12). It evinced that ⁶⁸Ga-PSMA PET/CT had an incredible ability to visualize bone metastases, with a superiority index of 7.3, which is higher than¹⁸F-NaF, ¹¹C-choline, ¹⁸F-choline, ¹⁸F-fluorodeoxyglucose (FDG), and ¹⁸F-fluciclovir PET/CT. Harmon et al. compared the application of ¹⁸F-PSMA-PET/CT and ¹⁸F-NaF in bone metastases, and 185 bone lesions were recognized by ¹⁸F-NaF and/or ¹⁸F-PSMA in 26 patients, in which ¹⁸F-NaF significantly works better (p<0.001) (13). Van Damme et al. conducted a study on 134 PCa patients, including newly diagnosed and relapsing patients, to make a comparison between PSMA imaging and whole-body magnetic resonance imaging (WB-MRI) for metastases diagnosis (14). PSMA imaging and WB-MRI were found to have no significant difference among identifying PCa patients with metastases when lymph node, skeletal, and visceral metastases were considered as a whole. However, in the subgroup of newly diagnosed PCa patients, PSMA PET/CT was better than WB-MRI for the detection of lymph node metastases, suggesting PSMA PET/CT is superior to WB-MRI for the recognition of lymph node metastasis in early PCa.

Although there are superior advantages of PSMA PET-CT imaging ascribed to the high expression of PSMA in PCa, some limitations exist. High uptake of radionuclide has been found in some benign lesions and other non-PCa malignant tumors in clinical applications (15, 16). In addition, the efficacy of PSMA PET/CT imaging is highly susceptible to the PSA level. The PSA level was positively associated with the SUV value of ¹⁸F-PSMA imaging in patients with androgen deprivation therapy (ADT) (13). Combining PSA levels and treatment status, ADT patients (n=11) with a PSA below 2 ng/ml showed more lesions on ¹⁸F-NaF than on ¹⁸F-PSMA (p=0.02). Among patients with PSA > 2 ng/ml, ADT patients (n=8) showed the same or more lesions on ¹⁸F-PSMA than on ¹⁸F-NaF. In efficacy monitoring, ¹⁸F-PSMA-1007-PET/CT has a good localization function for the biochemical recurrence (BCR) of PCa patients with a detection rate of 75% (17). Even small lymph node metastases less than 8 mm in diameter were imaged clearly. However, the remaining 25% of patients with a low level of PSA were not recognized by ¹⁸F-PSMA imaging. Similar to ¹⁸F-PSMA PET/CT in the relationship between the detection rate of BCR and PSA level, ⁶⁸Ga-PSMA PET/CT had this limitation in some studies (18, 19). Rauscher et al. analyzed subgroups of patients with very low (0.2-0.5 ng/ml) and low (0.5-1.0 ng/ml) PSA values and found that the detection rates of lesions in patients with recurred PCa were 55% (74/134) and 74% (102/138), respectively (18). Derlin et al. investigated the imaging efficacy of the ⁶⁸Ga-PSMA PET/CT with more PSA level stratifications and presented much lower detection rates in patients with low PSA (< 2 ng/ml) (19). However, this limitation could be overlooked in castration-resistant prostate cancer (CRPC), which is characterized by a rising PSA. Fourquet et al. performed PSMA-PET/CT imaging in incomplete CRPC patients, which were defined as non-metastatic PCa patients after ADT treatment (20). Even for patients with PSA serum levels less than 2 ng/ml, the positive rate of PSMA PET/CT imaging could reach 70%, suggesting the high effectiveness of PSMA PET/CT imaging for CRPC-relevant diseases.

In addition, PSMA PET/CT imaging works well with the prognosis of PCa. Liu et al. used ⁶⁸Ga-PSMA-617 PET/CT imaging semi-quantitative analysis indicators as “imaging markers” to predict risk stratification and metastasis risk of PCa (21). Univariate logistic regression models established by SUVmax, intraprostatic PSMA-derived tumor volume (iPSMA-TV), and intraprostatic total lesion PSMA (iTL-PSMA) could be able to efficiently previse high-risk PCa with the sensitivity and specificity of 87.5% and 50%, 62.5% and 100%, and 87.5% and 100%, respectively. A study performed by Roberts that included 848 patients after radical prostatectomy found that the SUVmax value of PSMA imaging lesions was remarkably negatively correlated with biochemical recurrence-free survival (BRFS) (22). Gleason score (GS) was also negatively correlated with BRF, and SUVmax value was an independent predictor of BRFS in patients. Roberts et al. found that increased ⁶⁸Ga-PSMA-11 uptake is often associated with poor pathological outcomes and provides prognostic information for progression-free survival (23). Changes in PSMA expression could be a predictive biomarker for overall survival, which may assist in personalizing therapy for PCa patients (24). ⁶⁸Ga-PSMA-11 PET/CT has a potential impact in guiding local lesion radiotherapy planning, which can improve the survival of castration-resistant PCa patients by adjusting the extent of radiotherapy (25). Under the guidance of PSMA PET, the mean time to PSA progression or last follow-up was 17.9 months with radiation therapy, compared with 2.9 months for patients without PSMA PET-guided local ablation radiation therapy (26). Shagera’s retrospective evaluation of 37 patients with metastatic hormone-sensitive or castration-resistant prostate cancer (mHSPC or mCRPC) by testing the biochemical association between responses and different PET parameters showed that ⁶⁸Ga-PSMA-11 PET/CT imaging could be an effective tool for evaluating the response of metastatic PCa to taxane chemotherapy (27).

In addition to the specific molecular markers mentioned above, G protein-coupled neurotensin receptor (NTR) and its ligand neurotensin peptide (NT) have been suggested to play an important role in PCa. Inhibiting the pathway of NTR1 has been suggested as a possible strategy to prevent the pathogenesis of this disease (28). Morgat et al. performed a pilot study of the NTR1 expression in 12 samples of normal prostate, 11 samples of benign prostatic hyperplasia (BPH), 44 samples of PCa, and 15 samples of metastatic lymph nodes (29). Compared with the negative NTR1 staining in normal prostate and BPH samples, 4 of the 44 primary tumors (9.1%) and 5 of 15 metastatic lymph nodes (33.3%) overexpressed NTR1, suggesting that NTR 1 may be a potential biomarker of PCa, especially for metastatic lymph nodes. However, the limited sample series seriously affects the reliability of this conclusion, and a larger sample size is needed in future studies.

Nevertheless, studies have suggested that NTR1 may be another molecular target that could complement PSMA imaging. Ma et al. developed novel heterodimeric probes that targeted both PSMA and NTR1 and showed significant uptake in both NTR1-positive/PSMA-negative PC-3 tumors and PSMA-positive/NTR1-negative LnCap tumors (two androgen-sensitive PCa xenografts) at the animal level (30). Zhang et al. used ⁶⁸Ga-DOTA-NT-20.3 animal PET imaging to scan the mice that were xenografted with PC-3, an androgen-receptor (AR)-positive, PCa cell line with no PSMA expression, suggesting that NTR1 may be a critical target for diagnosis or treatment of PCa applications with limited PSMA expression levels (31). However, the research of this tracer is still in the preclinical stage, and more preclinical and clinical studies are necessary for the exploration of its potential.

First described as a cell surface antigen F19 in 1986, fibroblast activation protein (FAP) is a 760 amino acid (AA)-glycoprotein and a member of the dipeptidyl peptidase (DPP) family (32, 33). FAP shares a high AA sequence homology with DPP4, leading to its high DPP activity (34). In addition, endopeptidase activity for cleavage of benzyloxycarbonyl-glycine-proline-7-amino-4-methylcoumarin was also found in FAP (33). Just as it initially caught people’s attention for its existence in the mesenchyme of multiple cancers rather than epithelial cells, FAP was found to be overexpressed in most epithelial cancers and participate in the regulation of tumor growth and metastasis, suggesting that FAP is a potential target for tumor theranostic (35, 36). Currently, FAP inhibitors (FAPI) are mainly used for FAP-targeted PET/CT imaging (37). In 2018, Loktev et al. first reported that ⁶⁸Ga-FAPI-02 was used for the imaging of multiple human malignant tumors and achieved good imaging results (38). Kratochwil et al. tested the ⁶⁸Ga-FAPI-04 on 80 patients with 28 kinds of tumors, in which PCa patients showed intermediate uptake of ⁶⁸Ga-FAPI-04 with SUV of 6-12 and TBR of 3-fold (39).

Kesch et al. developed tissue microarrays (TMAs) of prostate tissues from 94 PCa patients at various stages, including primary PCa, PCa receiving ADT, CRPC, and neuroendocrine prostate cancer (NEPC), with anti-FAP antibody staining, and found the positive correlation between FAP expression and disease advancement (40). The tissues with the highest FAP expression were from CRPC patients, suggesting the potential of FAPI imaging in advanced PCa, especially CRPC. A series of case studies for the FAPI PET/CT imaging on PSMA-negative CRPC also confirmed FAPI PET/CT imaging’s ability to visualize the metabolic lesions and complement the PSMA imaging (41–43). However, the issue of low sample size should be improved by large-scale clinical trials in the future.

The other weakness of FAPI PET/CT imaging is its specificity on tumor lesions. Xu et al. reported a case study of ⁶⁸Ga-DOTA-FAPI-04 on a PCa patient with arthritis. Compared with the prostate lesions, the arthritis site presented higher uptake of FAPI, suggesting that ⁶⁸Ga-DOTA-FAPI-04 may also be visualized in inflammation, possibly reducing its value in tumor diagnosis (44).

For tumor PET imaging, ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) is one of the most frequently used radiotracers. Fluorodeoxyglucose (FDG) is a glucose analogue, which is highly absorbed in tumor lesions mainly through glucose transporter-1 (GLUT1) because of its involvement in tumor cell metabolism. It has been broadly applicated in clinical diagnosis, staging analysis, prognosis prediction, and treatment response monitoring of various tumors as a PET imaging agent (45). However, some patients with well-differentiated PCa had false negatives during clinical imaging (46). In addition, some benign lesions, such as inflammation, can also take up ¹⁸F-FDG. Since the prostate is close to the bladder and ¹⁸F-FDG is mostly egested through the urinary tract, this limits its application in the primary tumor of PCa due to the bladder urinalysis activity (9).

Although ¹⁸F-FDG imaging possesses limited accuracy on primary PCa diagnosis and staging, high-grade PCa (GS= 8-10) and more aggressive metastatic PCa showed higher glycolytic activity. In a study of 148 PCa patients with biopsy GS ≥ 8, ¹⁸F-FDG PET/CT imaging detected lesions with high intraprostatic FDG uptake in 66% of patients (47). Intraprostatic FDG uptake was positively correlated with higher pathological GS, seminal vesicle invasion, pathological lymph node metastasis, and risk of BCR, suggesting that preoperative intraprostatic FDG uptake is a composite factor for poor pathological prognostic factors. In addition, ¹⁸F-FDG has a certain value in the detection of primary lesions of CRPC. Chen et al. studied 56 cases of CRPC with ⁶⁸Ga-PSMA and ¹⁸F-FDG PET/CT examinations (48). Although overall the ⁶⁸Ga-PSMA is significantly better than ¹⁸F-FDG PET/CT with a higher detection rate of 75.0% vs 51.8%, and more positive lesions of 135 vs 95, the incidence of patients with ⁶⁸Ga-PSMA−, ¹⁸F-FDG+ lesions was 23.2% (13/56), which could not be ignored in the clinic. The PSA level and GS of patients with ⁶⁸Ga-PSMA−, ¹⁸F-FDG+ lesions were higher than those of patients without ⁶⁸Ga-PSMA−, ¹⁸F-FDG+ lesions, that 61.5% of patients with GS ≥ 8 and PSA ≥ 7.9 ng/mL carried the special lesions, suggesting that CRPC patients with high GS and PSA may take advantage of ¹⁸F-FDG PET/CT imaging. ¹⁸F-FDG-PET/CT is also of great value in the diagnosis of bone metastases in high-grade PCa patients (GS≥8). In comparison with the bone scan, ¹⁸F-FDG PET/CT is sensitive and accurate in detecting bone metastases (sensitivity:100% vs 78.8%, specificity: 98.7% vs 98.2%) (49).

¹⁸F-FDG PET imaging also has the ability to assess prognosis in PCa patients. In the study of 94 patients with primary PCa who underwent ¹⁸F-FDG imaging previous to the radical prostatectomy, patients with higher SUVmax had poorer long-term survival (50). Higher intensity tracer uptake is positively associated with GLUT1 expression, stage, pathological grade, and disease progression. ¹⁸F-FDG PET whole-body total lesion glycolysis (TLG) is independently associated with overall survival as a quantitative prognostic imaging biomarker in mCRPC patients receiving abiraterone or enzalutamide as first-line therapy (51). Studies have shown that SUV value and the number of lesions are also independently associated with time to hormonal therapy failure (THTF). When the sum of SUVs was divided into quartile ranges, patients in the fourth quartile had significantly lower odds of survival than patients in the first quartile. Both SUV and ¹⁸F-FDG PET/CT-derived lesions provide independent prognostic information for THTF in patients with metastatic castration-sensitive PCa (52).

FDA approved the application of choline-based radiotracers (¹¹C and ¹⁸F- choline) in 2012 for patients with biochemically relapsed PCa. Now both ¹¹C and ¹⁸F- choline have been applied to monitor the curative effect in PCa patients. Wang et al. analyzed 46 studies and found that the combined sensitivity and specificity of ¹⁸F-choline for the detection of BCR of PCa were 0.93 (95% CI, 0.85-0.98) and 0.91 (95% CI, 0.73-0.97) (53). The combined detection rate was 66%, but when PSA is in the ranges of <0.5, 0.5-0.99, 1.0-1.99, and ≥2 ng/ml, the detection rates were 35%, 41%, 62%, 80%, respectively. Therefore, although the choline tracer is suitable for the detection of BCR of PCa, the detection rate is not ideal when the PSA value is very low.

¹¹C and ¹⁸F- choline also have implications in assessing prognosis in PCa. Jimbo et al. showed that ¹¹C-choline PET/CT assessment in mCRPC patients receiving primary docetaxel chemotherapy could predict overall treatment response and progression-free survival with blood pool-corrected SUVmax during treatment ( Figure 2 ) (54). The percent change in SUVmax was a significant predictor of complete response, with a greater than 20% reduction in SUVmax in 57 of 77 patients (74%), who were 3.6 times more likely to have complete remission than those patients with a reduction of SUVmax <20% after 6 cycles of primary docetaxel chemotherapy. Zhang et al. used ¹¹C-choline-PET to identify 89 patients with oligometastatic CRPC, providing a better target for stereotactic ablative radiotherapy (SABR) to improve the outcome with a median overall survival of 29.3 months (55). García Vicente et al. conducted interim and end-of-treatment ¹⁸F-Fluorocholine (FCH) PET/CT imaging in ²²³Ra-treated CRPC and bone metastases patients, and the results were significantly associated with both progression-free survival and overall survival, suggesting that interim and end-of-treatment ¹⁸F-FCH PET/CT imaging could be applied as predictors and even guidance during the ²²³Ra therapy (56).

¹⁸F-fluciclovine (¹⁸F-FACBC) was first reported by Shoup in 1999 for brain tumor imaging (57). Based on the encouraging diagnostic presentation and histologically confirmed data in patients with biochemical recurrence PCa, the FDA and European Commission (EC) approved ¹⁸F-FACBC for diagnostic imaging in PCa patients with elevated PSA after pre-treatment (58), and until recently, ¹⁸F-FACBC imaging has been included in the National Comprehensive Cancer Network (NCCN) guidelines for the management of BCR of PCa. A previous phase II clinical trial found the sensitivity and specificity of the scan to be 92.5% and 90.1%, respectively, for primary PCa lesions (59). Uptake of ¹⁸F-FACBC was significantly increased in PCa primary lesions, and lesions with high GS (>3+4) tended to show higher uptake rates compared with low GS lesions and benign prostatic hyperplasia (60). In the diagnosis of lymph node metastases, this study found that only 1 in 7 patients with metastatic lymph nodes showed true positive results on ¹⁸F-FACBC PET/CT and PET/MRI. Another multicenter phase II study of 40 regional lymph nodes in 28 patients found that the sensitivity, specificity, diagnostic accuracy, positive predictive value, and negative predictive value of ¹⁸F-FACBC imaging in lymph node analysis were 57.1% (4/7), 84.8% (28/33), 80.0% (32/40), 44.4% (4/9) and 90.3% (28/31), respectively (61). ¹⁸F-FACBC PET/CT imaging has no advantage in the diagnosis of bone metastases either, possibly due to the low spatial resolution and partial volume effects caused by necrotic and mucinous components in the metastatic foci (62). A meta-analysis included 9 studies and found that the pooled sensitivity and specificity of ¹⁸F-FACBC imaging of aged PCa patients (including both primary and recurrent PCa) were 86.3% and 75.9%, respectively, with a combined diagnostic odds ratio of 16.453 and heterogeneity of 30% (63). In the regional analysis, ¹⁸F-FACBC-PET/CT owned a higher sensitivity and a lower specificity for the assessment of tumors in the prostate bed than in the extraprostatic region (90.4% vs 76.5%, 89% vs 45%, respectively). Filippi et al. studied the clinical data of 81 patients who underwent ¹⁸F-FACBC PET/CT for BCR of PCa (64). The detection rate of ¹⁸F-FACBC PET/CT in the entire cohort accounted for 76.9%, and the positive predictive value was 96.7%. This modality played an impact on the clinical management in 33 of 81 patients (40.7%), resulting in a critical amendment in treatment strategy in 30 subjects (90.9%). Like PSMA imaging, the detection rate of FACBC imaging is positively correlated with the PSA levels. When the PSA levels are in the range of 0.2-0.57, 0.58-0.99, 1-1.5 and >1.5 ng/ml, the detection rates of ¹⁸F-FACBC PET/CT were 66.7%, 71.4%, 78.9% and 90, respectively. However, even at a low PSA level, ¹⁸F-FACBC PET/CT imaging preserves a much higher detection rate than PSMA imaging, which is meaningful for the localization and diagnosis of lesions and has a significant impact on clinical management.

Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor that is overexpressed in a variety of malignancies, such as breast cancer, PCa, and small cell lung cancer (65). GRPR is one of the subtypes of the bombesin (BBN) receptor, also called BB2r. As a bombesin analog, gastrin-releasing peptide (GRP) spreads over the peripheral nervous system and organs and primarily works in the gastrointestinal system through GRPR (66). As mentioned, the critical feature of GRPR is its overexpression in prostate tumor cells and underexpression in normal prostate tissue. Therefore, multiple radionuclides have been used to label bombesin analogs (GRPR agonists and antagonists), which preserve the high affinity for GRPR, to image tumors with high GRPR expressions (67, 68). At present, a variety of GRPR agonists and antagonists have emerged and been tagged with multiple radioisotopes. However, the GRPR agonists induce some gastrointestinal side effects due to the activation of GRPR. Compared with agonists, GRPR antagonists could provide better visualization with high value in the diagnosis and staging of PCa with less undesirable effects (69).

As one of the GRPR antagonists, RM26 was radiolabeled to trace the GRPR in prostate tumor tissues. In Zhang’s study, both NOTA-RM26 and agonist BBN were labeled with ⁶⁸Ga to image the lesions in 22 PCa patients (70). The results showed that the ⁶⁸Ga-RM26 tracer visualized much more primary lesions and metastases with significantly higher SUVmax than ⁶⁸Ga-BBN PET/CT ( Figure 3 ). Bakker et al. performed ⁶⁸Ga-SB3 PET/CT imaging on 10 PCa patients before radical resection with a sensitivity of 88% and a specificity of 88% in 16 lesions detected by prostatectomy pathology, suggesting that ⁶⁸Ga-SB3 PET/CT could be used for the detection and localization of primary PCa (71). Duan et al. compared ⁶⁸Ga-RM2 PET imaging with multiparametric magnetic resonance imaging (mpMRI) and ⁶⁸Ga-PSMA-11 PET on 41 patients with the initial diagnosis of intermediate and high-risk PCa. ⁶⁸Ga-RM2 and ⁶⁸Ga-PSMA11 had similar sensitivity and accuracy of 98%, 89% and 95%, 89%, respectively, which are significantly higher than mpMRI with 77% and 77%, for the detection of intraprostatic lesions (72). The post-prostatectomy histopathology also affirmed the ability of ⁶⁸Ga-RM2 PET imaging with a detection rate of 93%.

Not only for the initial diagnosis of PCa, but GRPR-targeted PET imaging could also take a role in the follow-up with the detection of BCR. Minamimoto et al. conducted a prospective study of 32 patients with BRC of PCa but negative imaging results on multiple conventional imaging modalities (CT, MRI, and ^99mTc-MDP bone scan) (73). Among the 32 participants, 23 individuals were recognized through the ⁶⁸Ga-RM2 PET imaging, suggesting a detection rate of 71.8% in these patients without positive findings on conventional imaging tools. Wieser et al. also collected 16 choline-PET/CT-negative/indeterminate biochemically recurrent PCa patients to evaluate the imaging ability of ⁶⁸Ga-RM2-PET/CT in detecting metastatic tumors and found that tumors in 10 out of 16 patients (62.5%) could be recognized by the ⁶⁸Ga-RM2-PET/CT imaging (74). In addition, the expression of GRPR appears to be unassociated with PSMA, suggesting that GRPR and PSMA-targeted PET imaging could be complementary (75). Therefore, GRPR-targeted imaging could complement other conventional modalities. Thus, GRPR tracer imaging is a promising tool for diagnosing and surveillance PCa with its high detection rate. However, the low sample size in these clinical trials critically affects the credibility of the evaluation of GRPR tracer imaging. More clinical trials with larger sample sizes are necessary for the future.

In conclusion, various imaging agents for the precision diagnosis of primary and metastatic PCa are under study, with both advantages and disadvantages ( Table 1 ). Although there is no 100% satisfactory imaging agent for PCa at present, with the in-depth research on current imaging agents and the development of new imaging agents, multi-target combined imaging or individualized imaging may bring better clinical value to PCa patients.

Indocyanine green (ICG), one of the most common near-infrared (NIR) fluorophores for fluorescence-guided surgery (FGS), has been approved by the FDA for more than 60 years. It is a 776 Da, amphiphilic tricarbocyanine, water-soluble, and anionic probe. It binds to protein quickly and is confined to the intravascular compartment through intravenous injection (76). The half-life of ICG is 150-180 seconds, and it has low toxicity. Glutathione S-transferase, a transport protein, is able to make ICG through the liver and excrete into bile totally; thus, ICG can be administered repeatedly every 15 minutes during surgery to label the tissue (77). Due to its relatively low cost and widespread availability, ICG is widely used in urologic surgery, including laparoscopic and robotic adrenalectomy procedures (78, 79). In laparoscopic robot-assisted radical prostatectomy (RARP), Mangano et al. used ICG with NIR fluorescence to guide the preservation of the neurovascular bundle (80). Tobis et al. adopted ICG to highlight the renal vasculature and distinguish between normal and malignant tissue (81). Rho et al. used CT to guide the penetration of ICG through fluorescence thoracoscopy, precise location and margin resection of the radiopaque lesions were confirmed via C-arm fluoroscopy, and pulmonary nodules were resected with an endostapler (82). As a result, the ICG imaging guided pulmonary nodule removal was 100% in the 24 patients. However, due to the nature that ICG is a non-targeted probe with suboptimal emission characteristics for NIR-II detection, it cannot distinguish between benign and malignant tumors and can be accumulated by other tissues, which may cause false positives (83). This disadvantage was shown by Tummers et al. in a study on oncologic procedures of fluorescence-guided surgery with a high false-positive rate (62%) for the application of ICG (84).

As mentioned, PSMA is a type II integral membrane glycoprotein that shows elevated expression in the majority of PCa cells (85). It is a marvelous target for image-based intraoperative guidance for accurate tumor identification due to three reasons. First, PSMA is exclusively overexpressed on tumor cells of primary PCa lesions, while its expression is consistently low in healthy prostate tissues. Second, the expression level of PSMA correlates with the Gleason grading of PCa lesions. Last, binding with the extracellular domain of PSMA normally induces internalization of the imaging agents, resulting in substantial retention of the labeling inside the tumor lesions (86). PSMA radio-guided surgery (PSMA-RGS) has been approved to be an efficient method for resecting primary tumors and metastatic lymph nodes (87).

Intravenous injection of ¹¹¹In-labeled PSMA-I&T to PCa patients during surgery has enabled the visualization of metastatic lymph nodes, which are normally unobtrusive and unrecognizable (88). Clinically, in patients undergoing salvage lymphadenectomy, the ¹¹¹In-PSMA-RGS allows intraoperative detection of small lymph node metastases with high specificity and sensitivity (89). In addition, the ¹¹¹In-PSMA-617 tracer also helped surgeons deal with unidentified pelvic lymph node metastases in situ during the surgery and resected ex vivo tissue samples to prove the successful removal (90). Except for ¹¹¹In-labeled PSMA ligands for detecting metastases of PCa, Robu et al. explored another ligand named ^99mTc-mas3-y-nal-k(Sub-KuE) for PCa imaging (91). Clinically, ^99mTc is preferable to ¹¹¹In, as it provides low-energy gamma rays that are more suitable for RGS due to the high sensitivity of gamma probes for collimation, and ^99mTc has a much shorter half-life (6 hours) than ¹¹¹In (2.8 days), resulting in faster pharmacokinetics and lower radiation exposure for both patients and nuclear medical professionals (92).

The hybrid tracer ICG-^99mTc-nanocolloid combining fluorescent dye ICG with the radioactive ^99mTc-nanocolloid, not only offers preoperative sentinel node (SN) mapping, but also provides better optical surgical guidance (93). The tracer shows no leakage into the surgical field and provides a depth estimation (>0.5–1 cm) of the nodal location, which helps to prevent surgery-related side effects (94). Another study also approved the value of the hybrid tracer in the surgical identification of lymph nodes (95). Overall, one obvious advantage of the ICG-^99mTc-nanocolloid tracer is that it can enable visualization of any tumor lesion or SN in their anatomical context during surgery, and its application is independent of the order of resection (primary tumor or metastasis) or the surgical setting (open or laparoscopic) (94).

Iatrogenic nerve injuries are common in prostatectomy, 20% of postoperative patients suffer from urinary incontinence, and many patients experience erectile dysfunction, which can only be partially mitigated by existing nerve-sparing surgical techniques. It is challenging to intraoperatively identify the specific location of buried small peripheral nerves (PNs), but the endeavor to find new ways to protect PNs is significant (96, 97). To meet the clinical need, an ideal method for imaging PNs during the intraoperative procedure should possess the following features. First, a high specificity and a good signal-to-noise ratio are essential. Second, real-time and long-term imaging is vital for PNs to be recognized and retained during surgery (98). Third, the imaging probes should have good biosafety. Last, the cost should be low enough for clinical use (97). Neurovascular dyes such as ICG and fluorescein have been used to highlight PNs in clinical settings (96). It has been shown that fluorescein was applied to visualize abnormal peroneal nerves in ganglion cyst excision procedures (99). Recently, ICG has been used to help protect critical functional structures in prostatectomy by enabling the identification of all neurovascular bundles without increasing the operative time or complications ( Figure 4 ) (80). These promising data indicate that iatrogenic injury can be prevented, and the operative time can be shortened with the help of fluorescence-guided imaging. According to the clinical study performed by Jin et al., in patients with ICG injected 24 hours prior to surgery, the pelvic autonomic nerves can be intraoperatively seen clearly under a NIR ray ( Figure 5 ) (100). Due to the ubiquity of such fluorophores, it is foreseeable that surgeons will attempt the fluorescent nerve-targeting agents more frequently in their clinical practice (96). However, the agents can have light penetrance through the tissue of greater than 5-6 mm. Such a deep penetration causes increased light scatter, thus obscuring the specific location of PNs. In addition, ICG is not a targeted dye, and it is not able to distinguish the nerve bundle from other tissues. For example, in the surgery for deep endometriosis, the ischemic lesion, the hypogastric nerve, the pelvic plexus, and the ureter were all dyed by ICG (101).

Extensive studies have reported that PCa tumor progression is favored by innervation. Magnon et al. reported that the formation of autonomic nerve fibers in the prostate gland regulates the development and dissemination of PCa (102). Therefore, biomarkers for innervation and effective visualization methods are necessary to assess nerve density in PCa. Nerve peptide 41 (NP41) has been found as a marker to highlight peripheral nerve tissue, and fluorescent-labeled NP41 can be visualized through its binding to the motor and sensory nerves in live mice (103, 104). Hingorani et al. reported that NP41 had the best nerve-to-non-nerve contrast compared to other peptides like NP38, 40, and 42, and the average nerve-to-non-nerve signal ratio increases by 17% under fluorescent imaging compared to white light (105). NP41 is considered an excellent agent for in vivo tracking of nerves in rodents. Since NP41 specifically targets nerves in PCa, it has the potential for visualizing nerve density and tumor innervation in PCa. The nanoprobes named propranolol-loaded-superparamagnetic iron oxide (SPIO)-NP41 nanoparticles (PSN NPs) have been used to assess the nerve density of PCa with high sensitivity and high specificity in mice (106). Since PSN NPs had an exclusive accumulation at the tumor site, benefiting the targeted delivery of propranolol, this study showed that PSN NPs inhibited PCa tumor growth by blocking the interaction between tumor cells and sympathetic nerves in the neural tumor microenvironment.

Nevertheless, existing data on applying NP41 to ex vivo human nerve tissue provided little contrast compared to muscle (105). Hence, human NP401 (HNP401), a peptide that binds to and highlights human autonomic and motor/sensory nerves, was identified for improving the labeling of human nerves, especially for the human prostate gland, suggesting its potential guidance role in the prostatectomy for PCa patients.